Antisense modulation of survivin expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of Survivin. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Survivin. Methods of using these compounds for modulation of Survivin expression and for treatment of diseases associated with expression of Survivin are provided.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/918,186 filedJul. 30, 2001, now U.S. Pat. No. 6,838,283 which is acontinuation-in-part of U.S. Ser. No. 09/496,694, filed Feb. 2, 2000,now U.S. Pat. No. 6,335,194 which is a continuation-in-part of U.S. Ser.No. 09/286,407 filed Apr. 5, 1999, issued as U.S. Pat. No. 6,165,788,which is a continuation-in-part of U.S. Ser. No. 09/163,162 filed Sep.29, 1998, issued as U.S. Pat. No. 6,077,709, all of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of Survivin. In particular, this invention relates toantisense compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding human Survivin. Sucholigonucleotides have been shown to modulate the expression of Survivin.

BACKGROUND OF THE INVENTION

A hallmark feature of cancerous cells is uncontrolled proliferation.Among the differences that have been discovered between tumor and normalcells is resistance to the process of programmed cell death, also knownas apoptosis (Ambrosini et al., Nat. Med., 1997, 3, 917-921). Apoptosisis a process multicellular organisms have evolved to preventuncontrolled cell proliferation as well as to eliminate cells that havebecome sick, deleterious, or are no longer necessary. The process ofapoptosis involves a multistep cascade in which cells are degraded fromwithin through the concerted action of proteolytic enzymes and DNAendonucleases, resulting in the formation of apoptotic bodies that arethen removed by scavenger cells. Research to date has shown that much ofthe intracellular degradation is carried out through the action of thecaspases, a family of proteolytic enzymes that cleave adjacent toaspartate residues (Cohen, Biochemistry Journal, 1997, 326, 1-16).

The finding that most tumor cells display resistance to the apoptoticprocess has led to the view that therapeutic strategies aimed atattenuating the resistance of tumor cells to apoptosis could represent anovel means to halt the spread of neoplastic cells (Ambrosini et al.,Nat. Med., 1997, 3, 917-921). One of the mechanisms through which tumorcells are believed to acquire resistance to apoptosis is byoverexpression of Survivin, a recently described member of the IAP(inhibitor of apoptosis) caspase inhibitor family. To date,overexpression of Survivin has been detected in tumors of the lung,colon, pancreas, prostate, breast, stomach, non-Hodgkin's lymphoma, andneuroblastoma (Adida et al., Lancet, 1998, 351, 882-883; Ambrosini etal., Nat. Med., 1997, 3, 917-921; Lu et al., Cancer Res., 1998, 58,1808-1812). A more detailed analysis has been performed in neuroblastomawhere it was found that Survivin overexpression segregated with tumorhistologies known to associate with poor prognosis (Adida et al.,Lancet, 1998, 351, 882-883). Finally, Ambrosini et al. describetransfection of HeLa cells with an expression vector containing a 708 ntfragment of the human cDNA-encoding effector cell protease receptor 1(EPR-1), the coding sequence of which is extensively complementary tothe coding strand of Survivin (Ambrosini et al., J. Bio. Chem., 1998,273, 11177-11182) and which potentially acts as a Survivin antisenseRNA. This construct caused a reduction in cell viability. Methods formodulating apoptosis and for reducing the severity of a pathologicalstate mediated by Survivin using agents that modulate amounts oractivity of Survivin are disclosed in WO 98/22589, which also disclosesthe EPR-1 coding strand/Survivin antisense construct described byAmbrosini et al., supra.

Survivin has recently been found to play a role in cell cycleregulation. It has been found to be expressed in the G2/M phase of thecell cycle in a cycle-regulated manner, and associates with microtubulesof the mitotic spindle. Disruption of this interaction results in lossof Survivin's anti-apoptotic function and increased caspase-3 activityduring mitosis. Caspase-3 is associated with apoptotic cell death. It istherefore believed that Survivin may counteract a default induction ofapoptosis in G2/M phase. It is believed that the overexpression ofSurvivin in cancer may overcome this apoptotic checkpoint, allowingundesired survival and division of cancerous cells. The Survivinantisense construct described by Ambrosini above was found todown-regulate endogenous Survivin in HeLa cells and to increasecaspase-3-dependent apoptosis in cells in G2/M phase. Li et al., Nature,1998, 396, 580-584.

As a result of these advances in the understanding of apoptosis and therole that Survivin expression is believed to play in conferring a growthadvantage to a wide variety of tumor cell types, there is a great desireto provide compositions of matter which can modulate the expression ofSurvivin. It is greatly desired to provide methods of diagnosis anddetection of nucleic acids encoding Survivin in animals. It is alsodesired to provide methods of diagnosis and treatment of conditionsarising from Survivin expression. In addition, improved research kitsand reagents for detection and study of nucleic acids encoding Survivinare desired.

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of Survivin. Consequently, there is a long-feltneed for agents capable of effectively inhibiting Survivin expression intumor cells. Antisense oligonucleotides against Survivin may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic andresearch applications.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularlyoligonucleotides, which are targeted to a nucleic acid encodingSurvivin, and which modulate the expression of Survivin. Pharmaceuticaland other compositions comprising the antisense compounds of theinvention are also provided. Further provided are methods of modulatingthe expression of Survivin in cells or tissues comprising contactingsaid cells or tissues with one or more of the antisense compounds orcompositions of the invention. Further provided are methods of treatingan animal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of Survivin byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

One embodiment of the present invention is a method of inhibiting theexpression of Survivin in human cells or tissues comprising contactinghuman cells or tissues with an antisense compound 8 to 30 nucleobases inlength targeted to a nucleic acid molecule encoding human survivin sothat expression of Survivin is inhibited.

The present invention also provides a method of treating an animalhaving a disease or condition associated with Survivin comprisingadministering to an animal having a disease or condition associated withSurvivin a therapeutically or prophylactically effective amount of anantisense compound 8 to 30 nucleobases in length targeted to a nucleicacid molecule encoding human survivin so that expression of Survivin isinhibited. Preferably, the disease or condition is a hyperproliferativecondition. In one embodiment, the hyperproliferative condition iscancer.

Another embodiment of the present invention is a method of treating ahuman having a disease or condition characterized by a reduction inapoptosis comprising administering to a human having a disease orcondition characterized by a reduction in apoptosis a prophylacticallyor therapeutically effective amount an of antisense compound 8 to 30nucleobases in length targeted to a nucleic acid molecule encoding humansurvivin so that expression of survivin is inhibited.

The present invention also provides a method of modulating apoptosis ina cell comprising contacting a cell with an antisense compound 8 to 30nucleobases in length targeted to a nucleic acid molecule encoding-humansurvivin so that apoptosis is modulated.

Still another embodiment of the invention is a method of modulatingcytokinesis in a cell comprising contacting a cell with an antisensecompound 8 to 30 nucleobases in length targeted to a nucleic acidmolecule encoding human survivin so that cytokinesis is modulated.

The present invention also provides a method of modulating the cellcycle in a cell comprising contacting a cell with an antisense compound8 to 30 nucleobases in length targeted to a nucleic acid moleculeencoding human survivin so that the cell cycle is modulated.

In still another embodiment of the invention, there is provided a methodof inhibiting the proliferation of cells comprising contacting cellswith an effective amount of an antisense compound 8 to 30 nucleobases inlength targeted to a nucleic acid molecule encoding human survivin, sothat proliferation of the cells-is inhibited. In one embodiment, thecells are cancer cells. The method may further comprise administering tothe patient a chemotherapeutic agent.

Preferably, the modulation of apoptosis is sensitization to an apoptoticstimulus. In one embodiment, the apoptotic stimulus is a cytotoxicchemotherapeutic agent. The method may further comprise contacting thecells with a chemotherapeutic agent. Preferably, the chemotherapeuticagent is taxol or cisplatin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding Survivin, ultimately modulating theamount of Survivin produced. This is accomplished by providing antisensecompounds which specifically hybridize with one or more nucleic acidsencoding Survivin. As used herein, the terms “target nucleic acid” and“nucleic acid encoding Survivin” encompass DNA encoding Survivin, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression ofSurvivin. In the context of the present invention, “modulation” meanseither an increase (stimulation) or a decrease (inhibition) in theexpression of a gene. In the context of the present invention,inhibition is the preferred form of modulation of gene expression andmRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding Survivin. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding Survivin, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 51-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′ UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′ UTR), known in the art to refer tothe portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA or correspondingnucleotides on the gene. The 5′ cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The 5′ cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease.

Aberrant fusion junctions due to rearrangements or deletions are alsopreferred targets. It has also been found that introns can also beeffective, and therefore preferred, target regions for antisensecompounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases.Particularly preferred are antisense oligonucleotides comprising fromabout 8 to about 30 nucleobases (i.e. from about 8 to about 30 linkednucleosides). Preferred embodiments comprise at least an 8-nucleobaseportion of a sequence of an antisense compound which inhibits expressionof Survivin. As is known in the art, a nucleoside is a base-sugarcombination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Representative United States patents thatteach the preparation of the above oligonucleosides include, but are notlimited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of whichis herein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH₂CH O₂CH₃ also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. patent applicationSer. No. 09/016,520, filed on Jan. 30, 1998, which is commonly ownedwith the instant application and the contents of which are hereinincorporated by reference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053, 5,639,873; 5,646,265;5,658,873; 5670,633; and 5,700,920, each of which is herein incorporatedby reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2EC (Sanghvi, Y. S.,Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferredbase substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is hereinincorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,73; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5;414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules.

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoc acid, naphthalene-2-sulfonicacid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis- and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of Survivin is treated by administering antisense compoundsin accordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingSurvivin, enabling sandwich and other assays to easily be constructed toexploit this fact.

Hybridization of the antisense oligonucleotides of the invention with anucleic acid encoding Survivin can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of Survivin in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal, intradermal and transdermal., oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection, drip or infusion; orintracranial, e.g., intrathecal or intraventricular, administration.Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1, 1-33).One or more penetration enhancers from one or more of these broadcategories may be included.

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651-654). Examples of some presentlypreferred fatty acids are sodium caprate and sodium laurate, used singlyor in combination at concentrations of 0.5 to 5%.

The physiological roles of bile include the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages934-935). Various natural bile salts, and their synthetic derivatives,act as penetration enhancers. Thus, the term “bile salt” includes any ofthe naturally occurring components of bile as well as any of theirsynthetic derivatives. Examples of presently preferred bile salts arechenodeoxycholic acid (CDCA) and/or ursodeoxycholic acid (UDCA),generally used at concentrations of 0.5 to 2%.

Complex formulations comprising one or more penetration enhancers may beused. For example, bile salts may be used in combination with fattyacids to make complex formulations. Preferred combinations include CDCAcombined with sodium caprate or sodium laurate (generally 0.5 to 5%).

Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA); citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 92-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; Buur et al., J.Control Rel., 1990, 14, 43-51). Chelating agents have the addedadvantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and perfluorochemical emulsions, such as FC-43 (Takahashi etal., J. Pharm. Pharmacol., 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, 8:2, 92-191); andnon-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

As used herein, “carrier compound” refers to a nucleic acid, or analogthereof, which is inert (i.e., does not possess biological activity perse) but is recognized as a nucleic acid by in vivo processes that reducethe bioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common-receptor. For example, the recovery of apartially phosphorothioated oligonucleotide in hepatic tissue is reducedwhen it is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

In contrast to a carrier compound, a “pharmaceutically acceptablecarrier” (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional compatible pharmaceutically-activematerials such as, e.g., antipruritics, astringents, local anestheticsor anti-inflammatory agents, or may contain additional materials usefulin physically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of theinvention.

Regardless of the method by which the antisense compounds of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of thecompounds and/or to target the compounds to a particular organ, tissueor cell type. Colloidal dispersion systems include, but are not limitedto, macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layer(s) made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech., 1995, 6, 698-708).

Certain embodiments of the invention provide for liposomes and othercompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide).

Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention. See,generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkowet al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49,respectively). Other non-antisense chemotherapeutic agents are alsowithin the scope of this invention. Two or more combined compounds maybe used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Examples of antisenseoligonucleotides include, but are not limited to, those directed to thefollowing targets as disclosed in the indicated U.S. patents, or pendingU.S. applications, which are commonly owned with the instant applicationand are hereby incorporated by reference, or the indicated published PCTapplications: raf (WO 96/39415, WO 95/32987 and U.S. Pat. Nos. 5,563,255and 5,656,612), the p120 nucleolar antigen (WO 93/17125 and U.S. Pat.No. 5,656,743), protein kinase C (WO 95/02069, WO 95/03833 and WO93/19203), multidrug resistance-associated protein (WO 95/10938 and U.S.Pat. No. 5,510,239), subunits of transcription factor AP-1 (pendingapplication U.S. Ser. No. 08/837,201, filed Apr. 14, 1997), Jun kinases(pending application U.S. Ser. No. 08/910,629, filed Aug. 13, 1997),MDR-1 (multidrug resistance glycoprotein; pending application U.S. Ser.No. 08/731,199, filed Sep. 30, 1997), HIV (U.S. Pat. Nos. 5,166,195 and5,591,600), herpesvirus (U.S. Pat. Nos. 5,248,670 and 5,514,577),cytomegalovirus (U.S. Pat. Nos. 5,442,049 and 5,591,720), papillomavirus(U.S. Pat. No. 5,457,189), intercellular adhesion molecule-1 (ICAM-1)(U.S. Pat. No. 5,514,788), 5-lipoxygenase (U.S. Pat. No. 5,530,114) andinfluenza virus (U.S. Pat. No. 5,580,767). Two or more combinedcompounds may be used together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-Alkoxy Amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham, Mass. orGlen Research, Inc. Sterling, Va.). Other 2′-O-alkoxy substitutednucleoside amidites are prepared as described in U.S. Pat. No.5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham, Mass.).

2′-Fluoro Amidites 2′-Fluorodeoxyadenosine Amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine.

Selective O-deacylation and triflation was followed by treatment of thecrude product with fluoride, then deprotection of the THP groups.Standard methodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.)

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/Acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside.

Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 hoursusing an overhead stirrer. POCl₃ was added dropwise, over a 30 minuteperiod, to the stirred solution maintained at 0-10° C., and theresulting mixture stirred for an additional 2 hours. The first solutionwas added dropwise, over a 45 minute period, to the latter solution. Theresulting reaction mixture was stored overnight in a cold room. Saltswere filtered from the reaction mixture and the solution was evaporated.The residue was dissolved in EtOAc (1 L) and the insoluble solids wereremoved by filtration. The filtrate was washed with 1×300 mL of NaHCO₃and 2×300 mL of saturated NaCl, dried over sodium sulfate andevaporated. The residue was triturated with EtOAc to give the titlecompound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100EC for 2 hours (tlc showed complete conversion). The vesselcontents were evaporated to dryness and the residue was dissolved inEtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (tlc showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-(Aminooxyethyl)nucleoside amidites and2′-(dimethylaminooxyethyl)nucleoside amidites

Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as per themethods of U.S. patent applications Ser. No. 10/037,143, filed Feb. 14,1998, and Ser. No. 09/016,520, filed Jan. 30, 1998, each of which iscommonly owned with the instant application and is herein incorporatedby reference.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hours), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference. 3′-Deoxy-3′-amino phosphoramidate oligonucleotides areprepared as described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me]chimeric phosphorothioate oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 Ammonia/Ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hours at roomtemperature is then done to deprotect all bases and sample was againlyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for24 hours at room temperature to deprotect the 2′ positions. The reactionis then quenched with 1M TEAA and the sample is then reduced to ½ volumeby rotovac before being desalted on a G25 size exclusion column. Theoligo recovered is then analyzed spectrophotometrically for yield andfor purity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]chimericphosphorothioate oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]chimericoligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55ECfor 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACEJ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACEJ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing four cell types are provided for illustrative purposes, butother cell types can be routinely used.

T-24 Cells:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5A basal media (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units permL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

3T3-L1 Cells:

The mouse embryonic adipocyte-like cell line 3T3-L1 was obtained fromthe American Type Culure Collection (Manassas, Va.). 3T3-L1 cells wereroutinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 80% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 4000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEMJ-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEMJ-1 containing 3.75 μg/mL LIPOFECTINJ(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium was replaced with freshmedium. Cells were harvested 16 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition of H-ras(for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as thescreening concentration for new oligonucleotides in subsequentexperiments for that cell line. If 80% inhibition is not achieved, thelowest concentration of positive control oligonucleotide that results in60% inhibition of H-ras or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of Survivin Expression

Antisense modulation of Survivin expression can be assayed in a varietyof ways known in the art. For example, Survivin mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISMJ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. Other methods of PCR are alsoknown in the art.

Survivin protein levels can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to Survivin can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total mRNA was isolated using an RNEASY 96J kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pippeting three times up anddown. The samples were then transferred to the RNEASY 96J well plateattached to a QIAVACJ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RWl was added to each well of the RNEASY 96J plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96J plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVACJ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVACJ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

Example 13 Real-Time Quantitative PCR Analysis of Survivin mRNA Levels

Quantitation of Survivin mRNA levels was determined by real-timequantitative PCR using the ABI PRISMJ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular (six-second) intervals bylaser optics built into the ABI PRISMJ 7700 Sequence Detection System.In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1×TAQMANJ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLDJ, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95EC toactivate the AMPLITAQ GOLDJ, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60EC for1.5 minutes (annealing/extension). Probes and primers to human Survivinwere designed to hybridize to a human Survivin sequence, using publishedsequence information (GenBank accession number U75285, incorporatedherein as SEQ ID NO:3). For human Survivin the PCR primers were:

-   forward primer: AAGGACCACCGCATCTCTACA (SEQ ID NO: 4)-   reverse primer: CCAAGTCTGGCTCGTTCTCAGT (SEQ ID NO: 5) and the PCR    probe was: FAM-CGAGGCTGGCTTCATCCACTGCC-TAMRA (SEQ ID NO: 6) where    FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent    reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.)    is the quencher dye. For human GAPDH the PCR primers were:-   forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)-   reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR    probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where    JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent    reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.)    is the quencher dye.

Probes and primers to mouse Survivin were designed to hybridize to amouse Survivin sequence, using published sequence information (GenBankaccession number AB013819, incorporated herein as SEQ ID NO: 10). Formouse Survivin the PCR primers were:

-   forward primer: CCGAGAACGAGCCTGATTTG (SEQ ID NO: 11)-   reverse primer: GGGAGTGCTTTCTATGCTCCTCTA (SEQ ID NO: 12) and the PCR    probe was: FAM-TAAGGAATTGGAAGGCTGGGAACCCG-TAMRA (SEQ ID NO: 13)    where FAM (PE-Applied Biosystems, Foster City, Calif.) is the    fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster    City, Calif.) is the quencher dye. For mouse GAPDH the PCR primers    were:-   forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)-   reverse primer: GGGTCTCGCTCCTGGAAGCT (SEQ ID NO: 15) and the PCR    probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID    NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is    the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,    Foster City, Calif.) is the quencher dye.

Example 14 Northern Blot Analysis of Survivin mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOLJ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBONDJ-N+nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using STRATALINKERJ UVCrosslinker 2400 (Stratagene, Inc, La Jolla Calif.) and then robed usingQUICKHYBJ hybridization solution (Stratagene, La Jolla, Calif.) usingmanufacturer's recommendations for stringent conditions.

To detect human Survivin, a human Survivin specific probe was preparedby PCR using the forward primer AAGGACCACCGCATCTCTACA (SEQ ID NO: 4) andthe reverse primer CCAAGTCTGGCTCGTTCTCAGT (SEQ ID NO: 5). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

To detect mouse Survivin, a mouse Survivin specific probe was preparedby PCR using the forward primer CCGAGAACGAGCCTGATTTG (SEQ ID NO: 11) andthe reverse primer GGGAGTGCTTTCTATGCTCCTCTA (SEQ ID NO: 12). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for mouse glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Example 15 Antisense Inhibition of Survivin Expression—PhosphorothioateOligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human Survivin RNA,using published sequences (GenBank accession number U75285, incorporatedherein as SEQ ID NO: 3). The oligonucleotides are shown in Table 1.Target sites are indicated by nucleotide numbers, as given in thesequence source reference (GenBank accession no. U75285), to which theoligonucleotide binds. All compounds in Table 1 areoligodeoxynucleotides with phosphorothioate backbones (internucleosidelinkages) throughout. All cytodines are 5-methylcytidines. The compoundswere analyzed for effect on Survivin mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments. If present “N.D.” indicates “no data”.

TABLE 1 Inhibition of human Survivin mRNA levels by phosphorothioateoligodeoxynucleotides SEQ ISIS TARGET % ID # REGION SITE SEQUENCEInhibition NO 23625 5′ UTR 1 gcgattcaaatctggcgg 0 17 23653 5′ UTR 19cctctgccaacgggtccc 4 18 23654 5′ UTR 75 tgagaaagggctgccagg 46 19 236555′ UTR 103 ttcttgaatgtagagatg 0 20 23656 5′ UTR 128 ggcgcagccctccaagaa38 21 23657 Coding 194 caagtctggctcgttctc 0 22 23658 Coding 226tccagctccttgaagcag 32 23 23659 Coding 249 ggtcgtcatctggctccc 36 24 23660Coding 306 gcttcttgacagaaagga 35 25 23661 Coding 323 ggttaattcttcaaactg0 26 23662 Coding 363 tcttggctctttctctgt 34 27 23663 Coding 393tcttattgttggtttcct 0 28 23664 Coding 417 tcgcagtttcctcaaatt 37 29 23665Coding 438 cgatggcacggcgcactt 72 30 23666 Coding 511 cctggaagtggtgcagcc16 31 23667 Coding 542 acaggaaggctggtggca 70 32 23668 Coding 587tttgaaaatgttgatctc 8 33 23669 Coding 604 acagttgaaacatctaat 0 34 23670Coding 625 ctttcaagacaaaacagg 0 35 23671 Coding 650 acaggcagaagcacctct 036 23672 Coding 682 aagcagccactgttacca 64 37 23673 Coding 700aaagagagagagagagag 18 38 23674 Coding 758 tccctcacttctcacctg 29 39 236753′ UTR 777 agggacactgccttcttc 43 40 23676 3′ UTR 808 ccacgcgaacaaagctgt62 41 23677 3′ UTR 825 actgtggaaggctctgcc 0 42 23678 3′ UTR 867aggactgtgacagcctca 62 43 23679 3′ UTR 901 tcagattcaacaggcacc 0 44 236803′ UTR 1016 attctctcatcacacaca 26 45 23681 3′ UTR 1054tgttgttaaacagtagag 0 46 23682 3′ UTR 1099 tgtgctattctgtgaatt 20 47 236833′ UTR 1137 gacttagaatggctttgt 37 48 23684 3′ UTR 1178cttgtctcctcatccacct 41 49 23685 3′ UTR 1216 aaaaggaqtatctgccag 39 5023686 3′ UTR 1276 gaggagcggccagcatgt 47 51 23687 3′ UTR 1373ggctgacagacacacggc 41 52 23688 3′ UTR 1405 ccgtgtggagaacgtgac 22 5323689 3′ UTR 1479 tacgccagacttcagccc 1 54 23690 3′ UTR 1514atgacagggaggagggcg 0 55 23691 3′ UTR 1571 gccgagatgacctccaga 66 56

As shown in Table 1, SEQ ID NOs: 19, 21, 23, 24, 25, 27, 29, 30, 32, 37,40, 41, 43, 48, 49, 50, 51, 52 and 56 demonstrated at least 30%inhibition of Survivin expression in this assay and are thereforepreferred.

Example 16 Antisense Inhibition of Survivin Expression—Phosphorothioate2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series ofoligonucleotides targeted to human Survivin were synthesized. Theoligonucleotide sequences are shown in Table 2. Target sites areindicated by nucleotide numbers, as given in the sequence sourcereference (GenBank accession no. U75285), to which the oligonucleotidebinds.

All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 18nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by four-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in otherexamples herein and are averaged from three experiments. If present,“N.D.” indicates “no data”.

TABLE 2 Inhibition of human Survivin mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapISIS SEQ # REGION TARGET SEQUENCE % ID 23692 5′ UTR 1 gcgattcaaatctggcgg22 57 23693 5′ UTR 19 cctctgccaacgggtccc 15 58 23694 5′ UTR 75tgagaaagggctgccagg 11 59 23695 5′ UTR 103 ttcttgaatgtagagatg 37 60 236965′ UTR 128 ggcgcagccctccaagaa 16 61 23697 Coding 194 caagtctggctcgttctc17 62 23698 Coding 226 tccagctccttgaagcag 0 63 23699 Coding 249ggtcgtcatctggctccc 19 64 23700 Coding 306 gcttcttgacagaaagga 35 65 23701Coding 323 ggttaattcttcaaactg 15 66 23702 Coding 363 tcttggctctttctctgt8 67 23703 Coding 393 tcttattgttggtttcct 41 68 23704 Coding 417tcgcagtttcctcaaatt 24 69 23705 Coding 438 cgatggcacggcgcactt 72 70 23706Coding 511 cctggaagtggtgcagcc 4 71 23707 Coding 542 acaggaaggctggtggca48 72 23708 Coding 587 tttgaaaatgttgatctc 2 73 23709 Coding 604acagttgaaacatctaat 28 74 23710 Coding 625 ctttcaagacaaaacagg 0 75 23711Coding 650 acaggcagaagcacctct 38 76 23712 Coding 682 aagcagccactgttacca27 77 23713 Coding 700 aaagagagagagagagag 0 78 23714 Coding 758tccctcacttctcacctg 0 79 23715 3′ UTR 777 agggacactgccttcttc 44 80 237163′ UTR 808 ccacgcgaacaaagctgt 25 81 23717 3′ UTR 825 actgtggaaggctctgcc8 82 23718 3′ UTR 867 aggactgtgacagcctca 49 83 23719 3′ UTR 901tcagattcaacaggcacc 0 84 23720 3′ UTR 1016 attctctcatcacacaca 0 85 237213′ UTR 1054 tgttgttaaacagtagag 0 86 23722 3′ UTR 1099 tgtgctattctgtgaatt80 87 23723 3′ UTR 1137 gacttagaatggctttgt 44 88 23724 3′ UTR 1178ctgtctcctcatccacct 27 89 23725 3′ UTR 1216 aaaaggagtatctgccag 21 9023726 3′ UTR 1276 gaggagcggccagcatgt 39 91 23727 3′ UTR 1373ggctgacagacacacggc 45 92 23728 3′ UTR 1405 ccgtgtggagaacgtgac 24 9323729 3′ UTR 1479 tacgccagacttcagccc 25 94 23730 3′ UTR 1514atgacagggaggagggcg 0 95 23731 3′ UTR 1571 gccgagatgacctccaga 19 96

As shown in Table 2, SEQ ID NOs: 60, 65, 68, 70, 72, 76, 80, 83, 87, 88,91 and 92 demonstrated at least 30% inhibition of Survivin expression inthis experiment and are preferred.

Example 17 Antisense Inhibition of Survivin Expression—Phosphorothioate2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a third series ofoligonucleotides targeted to human Survivin mRNA were synthesized. Theoligonucleotide sequences are shown in Table 3. Target sites areindicated by nucleotide numbers to which the oligonucleotide binds. Thehuman Survivin mRNA was generated by splicing nucleotides 2811-2921,3174-3283, 5158-5275 and 11955-12044 from GenBank accession no. U75285creating the complete human mRNA sequence herein incorporated as SEQ IDNO: 97.

All compounds in Table 3 are chimeric oligonucleotides (“gapmers”) 18nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by four-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in otherexamples herein and are averaged from three experiments. If present,“N.D.” indicates “no data”.

TABLE 3 Inhibition of human Survivin mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapSEQ ISIS TARGET % ID # REGION SITE SEQUENCE Inhibition NO 107289 Coding14 gccaacgggtcccgcgat 5 98 107290 Coding 35 catgccgccgccgccacc 4 99107291 Coding 90 agatgcggtggtccttga 94 100 107292 Coding 110gggccagttcttgaatgt 14 101 107293 Coding 166 tggatgaagccagcctcg 0 102107294 Coding 212 gcagaagaaacactgggc 0 103 107295 Coding 233ccagccttccagctcctt 0 104 107296 Coding 283 caaccggacgaatgcttt 0 105107297 Coding 299 gacagaaaggaaagcgca 83 106 107298 Coding 313tcaaactgcttcttgaca 73 107 107299 Coding 329 accaagggttaattcttc 0 108107300 Coding 359 ggctctttctctgtccag 7 109 107301 Coding 370attttgttcttggctctt 4 110 107302 Coding 398 tttcttcttattgttggt 11 111107303 Coding 412 gtttcctcaaattctttC 0 112 107304 Coding 421ttcttcgcagtttcctca 49 113 107305 Coding 432 cacggcgcactttcttcg 22 114107306 Coding 445 agctgctcgatggcacgg 7 115 107307 Coding 495ccactctgggaccaggca 0 116 107308 Coding 514 aaccctggaagtggtgca 0 117107309 Coding 529 tggcaccagggaataaac 0 118 107310 Coding 566tcctaagacattgctaag 1 119 107311 Coding 579 tgttgatctcctttccta 3 120107312 Coding 590 taatttgaaaatgttgat 15 121 107313 Coding 599tgaaacatctaatttgaa 0 122 107314 Coding 613 aacaggagcacagttgaa 27 123107315 Coding 619 agacaaaacaggagcaca 0 124 107316 Coding 630tgccactttcaagacaaa 24 125 107317 Coding 635 tctggtgccactttcaag 0 126107318 Coding 653 Tgcacaggcagaagcacc 15 127 107319 Coding 676ccactgttaccagcagca 4 128 107320 Coding 701 aaaagagagagagagaga 0 129107321 Coding 766 cttcttcctccctcactt 7 130 107322 Coding 789agctctagcaaaagggac 0 131 107323 Coding 814 ctctgcccacgcgaacaa 13 132107324 Coding 836 cagacacattcactgtgg 0 133 107325 Coding 852tcaacaacatgaggtcca 0 134 107326 Coding 882 gccaagtccacactcagg 0 135107327 Coding 1039 gaggagccagggactctg 16 136 107328 Coding 1067aataagaaagccatgttg 0 137 107329 Coding 1080 acaattcaaacaaaataa 30 138107330 Coding 1081 aacaattcaaacaaaata 0 139 107331 Coding 1082taacaattcaaacaaaat 3 140 107332 Coding 1083 ttaacaattcaaacaaaa 31 141107333 Coding 1084 attaacaattcaaacaaa 9 142 107334 Coding 1085aattaacaattcaaacaa 10 143 107335 Coding 1092 ttctgtgaattaacaatt 16 144107336 Coding 1093 attctgtgaattaacaat 0 145 107337 Coding 1094tattctgtgaattaacaa 25 146 107338 Coding 1095 ctattctgtgaattaaca 12 147107339 Coding 1096 gctattctgtgaattaac 14 148 107340 Coding 1097tgctattctgtgaattaa 14 149 107341 Coding 1098 gtgctattctgtgaatta 8 150107342 Coding 1100 ttgtgctattctgtgaat 18 151 107343 Coding 1101tttgtgctattctgtgaa 33 152 107344 Coding 1102 gtttgtgctattctgtga 11 153107345 Coding 1103 agtttgtgctattctgtg 21 154 107346 Coding 1104tagtttgtgctattctgt 17 155 107347 Coding 1105 gtagtttgtgctattctg 57 156107348 Coding 1106 tgtagtttgtgctattct 6 157 107349 Coding 1107ttgtagtttgtgctattc 13 158 107350 Coding 1108 attgtagtttgtgctatt 15 159107351 Coding 1109 aattgtagtttgtgctat 0 160 107352 Coding 1110taattgtagtttgtgcta 25 161 107353 Coding 1120 tgcttagttttaattgta 0 162107354 Coding 1144 ccccaatgacttagaatg 7 163 107355 Coding 1163cctgaagttcaccccgtt 19 164 107356 Coding 1184 tctattctgtctcctcat 0 165107357 Coding 1199 gacgcttcctatcactct 18 166 107358 Coding 1222agtggcaaaaggagtatc 0 167 107359 Coding 1239 ctgtctaatcacacagca 0 168107360 Coding 1281 tgagggaggagcggccag 0 169 107361 Coding 1350gcagcccagccagtcccc 0 170 107362 Coding 1379 aggttgggctgacagaca 1 171107363 Coding 1399 ggagaacgtgacagatgt 23 172 107364 Coding 1425gggcggactgcgtctctc 0 173 107365 Coding 1470 cttcagccctgcgggagc 0 174107366 Coding 1488 ccatcatcttacgccaga 0 175 107367 Coding 1509agggaggagggcgaatca 0 176 107368 Coding 1585 atttctcaggaacagccg 7 177

As shown in Table 3, SEQ ID NOs: 101, 106, 107, 113, 138, 141, 152 and156 demonstrated at least 30% inhibition of human Survivin expression inthis assay and are preferred.

Example 18 Antisense Inhibition of Mouse Survivin Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap.

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the mouse Survivin RNA,using published sequences (GenBank accession number AB013819,incorporated herein as SEQ ID NO: 10). The oligonucleotides are shown inTable 4. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 4 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on mouse Survivin mRNA levels by quantitative real-time PCRas described in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 4 Inhibition of mouse Survivin mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapSEQ ISIS TARGET % ID # REGION SITE SEQUENCE Inhibition NO 114968 5′ UTR3 agagccccggccccctcgtg 0 178 114967 5′ UTR 4 gagagccccggccdcctcgt 0 179114966 5′ UTR 16 agagcatgccgggagagccc 0 108 114965 5′ UTR 25gcgcgccgcagagcatgccg 0 181 114964 5′ UTR 55 aaacgcaggattcaaatcgc 0 182114963 5′ UTR 66 caagacgactcaaacgcagg 0 183 114962 5′ UTR 68gccaagacgactcaaacgca 0 184 114961 Start 92 catgatggcgtcaccacaac 0 185Codon 114972 Start 101 cggagctcccatgatggcgt 27 186 Codon 114960 Start104 cgccggagctcccatgatgg 47 187 Codon 114959 Coding 171ggaagggccagttcttgaag 35 188 114958 Coding 184 gcgcagtcctccaggaaggg 0 189114957 Coding 186 aggcgcagtcctccaggaag 10 190 114957 Coding 186aggcgcagtcctccaggaag 6 191 114971 Coding 189 tgcaggcgcagtcctccagg 30 192114956 Coding 249 aatcaggctcgttctcggta 46 193 114955 Coding 259cactgggccaaatcaggctc 14 194 114954 Coding 289 cagccttccaattccttaaa 0 195114953 Coding 300 catcgggttcccagccttcc 67 196 114952 Coding 303tgtcatcgggttcccagcct 83 197 114951 Coding 315 cctctatcgggttgtcatcg 40198 114950 Coding 327 gctttctatgctcctctatc 39 199 114949 Coding 358ttgacagtgaggaaggcgca 0 200 114948 Coding 374 ttcttccatctgcttcttga 0 201114947 Coding 387 cactgacggttagttcttcc 39 202 114946 Coding 389ttcactgacggttagttctt 12 203 114945 Coding 394 aagaattcactgacggttag 26204 114944 Coding 396 tcaagaattcactgacggtt 38 205 114943 Coding 465cttcaaactctttttgcttg 10 206 114942 Coding 497 ctcaattgactgacgggtag 48207 114941 Coding 498 gctcaattgactgacgggta 39 208 114940 Coding 499tgctcaattgactgacgggt 23 219 114939 Stop 521 ggctcagcattaggcagcca 18 210114938 Stop 531 tctcagcaaaggctcagcat 42 211 Codon 114937 3′ UTR 601gctaggaggccctggctgga 52 212 114936 3′ UTR 613 ctctaagatcctgctaggag 39213 114935 3′ UTR 627 accactgtctccttctctaa 35 214 114934 3′ UTR 642atccagtttcaaaataccac 0 215 114933 3′ UTR 649 atttgatatccagtttcaaa 20 216114932 3′ UTR 666 aaagcaaaaccaaaaatatt 7 217 114931 3′ UTR 683agagaggtagccactttaaa 45 218 114930 3′ UTR 688 accaaagagaggtagccact 44219 114929 3′ UTR 713 cgtcacaatagagcaaagcc 14 220 114970 3′ UTR 721taagtccacgtcacaataga 7 221 114928 3′ UTR 741 ttcatcacttccttattgct 8 222114927 3′ UTR 756 agagaacactgtcccttcat 15 223 114969 3′ UTR 786acaggcaccccgacccccac 4 224 114926 3′ UTR 801 gaaccaagaccttgcacagg 59 225114925 3′ UTR 812 tatcacaatcagaaccaaga 34 226 114924 3′ UTR 834cattagcagccctgtatgga 18 227 114923 3′ UTR 856 aaccacacttacccatgggc 52228 114922 3′ UTR 903 gtggtaggaaaactcatcag 64 229 114921 3′ UTR 934actttttcaagtgattttat 13 230

As shown in Table 4, SEQ ID NOs: 187, 188, 192, 193, 196, 197, 198, 199,202, 205, 207, 208, 211, 212, 213, 214, 218, 219, 225, 226, 228 and 229demonstrated at least 30% inhibition of mouse Survivin expression inthis experiment and are therefore preferred.

In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseSurvivin RNA, using published sequences (GenBank accession numberAA717921, incorporated herein as SEQ ID NO: 231). The oligonucleotidesare shown in Table 5. “Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which theoligonucleotide binds. All compounds in Table 5 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseSurvivin mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments. If present,“N.D.” indicates “no data”.

TABLE 5 Inhibition of mouse Survivin mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapSEQ ISIS TARGET % ID # REGION SITE SEQUENCE Inhibition NO 114920 5′ UTR2 aatcccagccaaggatccga 0 232 114919 5′ UTR 21 cgtggtggctcacaccttta 1 233114918 5′ UTR 33 tttcaagccgggcgtggtgg 11 234 114917 5′ UTR 57acatatatatatataaacat 0 235 114916 5′ UTR 87 aattttccttccttgatttt 5 236114915 5′ UTR 105 tactgagctacaaactggaa 41 237 114914 5′ UTR 108acttactgagctacaaactg 0 238 114913 5′ UTR 168 aagttattatttttgtattg 0 239114912 5′ UTR 169 aaagttattatttttgtatt 7 240 114911 5′ UTR 184taaatcattaaaaggaaagt 0 241 114910 5′ UTR 197 catcgtggcaagataaatca 0 242114909 5′ UTR 229 gcctgtccagggtgagatgc 0 243 114908 5′ UTR 231ttgcctgtccagggtgagat 0 244 114907 5′ UTR 240 gggccaggcttgcctgtcca 13 245114906 Start 293 ggtctcctttgcctggaatg 23 246 114905 Start 296gttggtctcctttgcctgga 59 247 Codon

As shown in Table 5, SEQ ID NOs: 237 and 247 demonstrated at least 30%inhibition of mouse Survivin expression in this experiment and aretherefore preferred.

Example 19 Western Blot Analysis of Survivin Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 hours after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to Survivin is used,with a radiolabelled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale, Calif.).

Example 20 Effect of Antisense Inhibition of Survivin on Apoptosis

ISIS 23722 and a mismatch control, ISIS 28598 (TAAGCTGTTCTATGTGTT; SEQID NO: 248) were assayed for their effect on apoptosis in HeLa cells.The caspase inhibitor z-VAD.fmk was purchased from Calbiochem (La Jolla,Calif.) and used according to manufacturer's recommendations. In HeLacells without oligonucleotide, approximately 4% of cells are hypodiploid(indicating DNA fragmentation, a measure of apoptosis). With theaddition of ISIS 23722, approximately 22% of cells are hypodiploid,compared to approximately 11% with the mismatch oligonucleotide. In thepresence of the caspase inhibitor z-VAD.fmk (42.8 mM), the percent ofhypodiploid (apoptotic) cells drops to 3% without oligonucleotide, 6%with ISIS 23722 and 4% with the mismatch control. This demonstrates thatantisense inhibition of Survivin increases apoptosis and that thiseffect is caspase-mediated.

Example 21 Effect of Antisense Inhibition of Survivin on Cytokinesis

HeLa cells treated with an antisense oligonucleotide targeted toSurvivin (ISIS 23722) can be observed to form large, multinucleatedcells as a result of improper cell division. The mismatch controloligonucleotide did not have this effect and cells appeared normal(comparable to untreated controls). This effect can be quantitated byflow cytometry.

Untreated cells or cells treated with the control oligonucleotidedisplay two prominent peaks, representing populations of cells in the G1phase and the G2/M phase of cell division, respectively. G1 cells have asingle copy of their DNA (1×) and G2/M cells have two copies (2×). Overtime from 24 hours to 72 hours, these 1× and 2× peaks remain virtuallyunchanged in cells treated with the control oligonucleotide or withoutoligonucleotide. However, in cells treated with the antisenseoligonucleotide targeted to Survivin, the majority of cells have twocopies of DNA by 24 hours after oligo treatment. This indicates thatcell division is arrested. By 48 hours after treatment with thisoligonucleotide, a 4× peak is approximately equal in size to the 1× and2× peaks, indicating roughly equal numbers of cells with one, two andfour copies of DNA. By 72 hours the largest peak is 16×, indicating thatcells have 16 copies of their DNA and thus that division of thecytoplasm has not occurred for multiple generations. Thus inhibition ofSurvivin is shown to interfere with cytokinesis.

Example 22 Effect of Antisense Inhibition of Survivin on CellProliferation

Human HT1080 fibrosarcoma cells (American Type Culture Collection,CCL-121) were grown in minimal essential medium with 1% non-essentialamino acids, 90% with 10% fetal bovine serum (Gibco BRL). Cells wereelectroporated (Electro Square Porator, Model T820, Biotechnologies andExperimental Research, BTX) with oligonucleotide at settings of 225volts for 6 milliseconds with a single pulse and oligonucleotideconcentrations of 1 to 30 μM. ISIS 23722 (SEQ ID NO: 87) and themismatch control ISIS 28598 (SEQ ID NO: 248) were used. Cells wereplated at 1500 cells/well immediately after electroporation and viablecells were measured by MTT assay at 24, 48, 72, 96 and 120 hours afterelectroporation. Growth rate (OD/hour) was plotted againstoligonucleotide concentration. At an oligonucleotide concentration of 1μM, growth rates were virtually identical for ISIS 23722 and thecontrol, ISIS 28598 (0.01726 and 0.01683, respectively. At 5 μMoligonucleotide, the growth rate of the ISIS 23722-treated cells was16.7% less than the control treated cells (0.01433 vs. 0.01728 ?OD/hour,respectively). At 10 μM the growth rate of the ISIS 23722-treated cellswas 45% less than the control treated cells (0.009677 vs. 0.01762?OD/hour, respectively). At 20 μM the growth rate of the ISIS23722-treated cells was 52% less than the control treated cells(0.007716 vs. 0.01620 OD/hour, respectively). At 30 μM the growth rateof the ISIS 23722-treated cells was 54% less than the control treatedcells (0.006562 vs. 0.01417 OD/hour, respectively). Thus treatment withantisense oligonucleotide targeted to Survivin was demonstrated toreduce the rate of tumor cell proliferation by over 50%.

In an similar experiment using a different control oligonucleotide, a20mer random oligonucleotide (ISIS 29848, SEQ ID NO: 249;NNNNNNNNNNNNNNNNNNNN wherein each N is a mixture of A, C, G and T) asimilar result was obtained. Oligonucleotides were tested atconcentrations of 0.5 to 20 μM, and cell viability was again measured byMTT assay and growth rate (OD/hour) was calculated. At 0.5 μMoligonucleotide concentrations, growth rates were similar for ISIS 23722and control treated cells (0.01441 and 0.01342, respectively). At 10 μMthe growth rate of the ISIS 23722-treated cells was 57% less than thecontrol treated cells (0.005568 vs. 0.01298 OD/hour, respectively). At20 μM the growth rate of the ISIS 23722-treated cells was 77% less thanthe control treated cells (0.002433 vs. 0.01073 OD/hour, respectively).Thus treatment with antisense oligonucleotide targeted to Survivin wasdemonstrated to reduce the rate of tumor cell proliferation by over 75%compared to control.

A similar experiment was conducted in human MCF-7 breast carcinomacells, testing ISIS 23722 and the random control ISIS 29848 at dosesfrom 0.5 to 20 μM. Cells were electroporated (Electro Square Porator,Model T820 manufactured by Biotechnologies and Experimental Research,BTX) at setting as of 175 volts for 6 milliseconds with a single pulsewith oligonucleotide and growth rates were calculated as describedabove. At 0.5 μM oligonucleotide concentrations, growth rates weresimilar for ISIS 23722 and control treated cells (0.005959 and 0.005720,respectively). At 1 μM oligonucleotide, growth rates were stillrelatively similar for ISIS 23722 and control treated cells (0.005938and 0.005479, respectively). At 5 μM oligonucleotide, growth rates were0.002574 and 0.005676, respectively for ISIS 23722 and control treatedcells. At 10 μM the growth rate of the ISIS 23722-treated cells was 69%less than the control treated cells (0.001828 vs. 0.005901 OD/hour,respectively). At 20 μM the growth rate of the ISIS 23722-treated cellswas 64% less than the control treated cells (0.001523 vs. 0.004223OD/hour, respectively). Thus treatment with antisense oligonucleotidetargeted to Survivin was demonstrated to significantly reduce the rateof tumor cell proliferation in several tumor cell types.

Example 23 Sensitization of Cells to Chemotherapeutic Agent Stimuli byISIS 23722

ISIS 23722 (SEQ ID NO: 87) and a control oligonucleotide, ISIS 29848, a20mer random oligonucleotide (ISIS 29848, SEQ ID NO: 249;NNNNNNNNNNNNNNNNNNNN wherein each N is a mixture of A, C, G and T) wereassayed for their ability to sensitize cells to the effects of thechemotherapeutic agents, Taxol and Cisplatin.

Human HT1080 fibrosarcoma cells (American Type Culture Collection,CCL-121) were grown in minimal essential medium with 1% non-essentialamino acids, 90% with 10% fetal bovine serum (Gibco BRL). Cells weretreated with oligonucleotide at concentrations of 10 to 100 nM alone orin combination with Taxol (concentrations of 0.25 nM or 1 nM) orCisplatin (concentrations of 5 μM or 25 μM). Treatment with Taxol orCisplatin followed oligonucleotide treatment by 1-2 hr. Cells wereplated at 1500 cells/well immediately after treatment and viable cellswere measured by MTT assay at 12, 24, 36, 48, and 60 hours aftertreatment. Growth rate (OD/hour) is plotted against oligonucleotideand/or chemotherapeutic agent concentration.

A similar experiment was conducted in human MCF-7 breast carcinoma cells(American Type Culture Collection), testing ISIS 23722 and the randomcontrol ISIS 29848 at doses from 10 to 100 nM alone or in combinationwith Taxol (concentrations of 0.5 nM or 2 nM) or Cisplatin(concentrations of 2.5 μM or 15 μM). Cells were grown in Dulbecco'sModified Eagles medium (low glucose), 90% with 10% fetal bovine serum(Gibco BRL). Treatment with Taxol or Cisplatin followed oligonucleotidetreatment by 1-2 hr. Cells were plated at 2500 cells/well immediatelyafter transfection and viable cells were measured by MTT assay at 12,24, 36, 48, and 60 hours after treatment. Growth rate (OD/hour) isplotted against oligonucleotide and/or chemotherapeutic agentconcentration.

Example 24 Mixed Backbone Version of Active Oligonucleotide ISIS 23722

An oligonucleotide having the same sequence as ISIS 23722 (SEQ ID NO:87)was synthesized, this time as a 2′ MOE gapmer with phosphodiesterbackbone linkages in the 2′MOE “wings” and phosphorothioate linkages inthe 2′ deoxy “gap”. Both cytosines are 5-methylcytosines.

This compound is tested for its effects on cell proliferation,cytokinesis and sensitization to chemotherapeutic agents as describedherein in previous examples.

Example 25 Down-Regulation of IL-11-Induced Survivin Expression on HumanSkin Engrafted onto Immunodeficient Mice

The efficacy of anti-survivin antisense oligonucleotide cream todown-regulate IL-11 induced survivin expression in human skin grafts wasexamined in immunodeficient mice. SCID/beige mice were engrafted withhuman skin obtained from elective surgery and allowed to heal for 5weeks. Four mice with 2 skin grafts each were used. The right graft ofeach animal received control (placebo) antisense oligonucleotide cream,while the left graft received the anti-survivin antisenseoligonucleotide cream which comprised 5% of the 2′-MOE mixed backbonedeoxyoligonucleotide ISIS 28599 (5′-TGTGCTATTCTGTGAATT-3′, SEQ ID NO:250) (nucleotides 1-4 and 15-18 are 2′-MOE; cytosines at positions 5 and10 are 5-meC; nucleotides 1-3 and 16-18 are phosphodiester linkages;nucleotides 4-15 are phosphorothioate linkages. The dosing scheduleconsisted of regular dosing of 100 μl/graft 3 times each day for 3 days.The morning of the 4^(th) day, skin grafts were given their morning dosethen 1 hour later injected intradermally with recombinant human IL-11(500 ng) (Genetics Institute, Andover, Mass.). Antisense oligonucleotidecream was administered for 2 final doses. Animals were sacrificed on themorning of day 5. Grafts were harvested and formalin fixed for paraffinembedding.

Grafts from one animal were not identified in the blocks. The remainingthree animals were evaluated by immunostaining for survivin and byroutine hematoxylin and eosin histology. Immunostaining was performed ondeparaffinized sections as previously described. Briefly, 5-μm tissuesections were cut and slides were baked overnight in a 60° C. oven.Sections were deparaffinized in xylene for 5 hours, washed twice inethanol, and endogenous peroxidase activity was quenched with 1.5%hydrogen peroxide in methanol for 10 min. Slides were then placed in a 4quart Wear-Ever pressure cooker (Mirro Co., Manitowoc, Wis.) containing1.5 liters of 9 mM sodium citrate, pH 6.0 that had been brought to aboil. The lid was secured, and heating was continued (approx. 6 min)until the pressure valve released. Slides were gently cooled by fillingthe pressure cooker with tap water, and then washed 3 times with waterand once with PBS, pH 7.0. Staining was performed using a primary rabbitpolyclonal antibody against survivin at a concentration of 0.1 μg/ml inPBS, pH 7.0, containing 0.5% bovine serum albumin (BSA) and 5% normalgoat serum (Vector laboratories) and incubated overnight at 4° C. usinga Histostain-Plus kit (Zymed) with 3,3′-diaminobenzidine (DAB) as thechromogen.

The histology appeared normal and indistinguishable between the treatedand control groups. Survivin expression showed a downregulation ofexpression in grafts treated with the survivin antisense oligonucleotidecream compared to controls. Both the dermal microvessels and theepidermal keratinocytes showed this downregulation when compared tocontrol grafts.

1. A modified antisense compound comprising the nucleotide sequenceshown in SEQ ID NO:87, or a pharmaceutically acceptable salt thereof. 2.A modified or unmodified antisense compound consisting of the nucleotidesequence shown in SEQ ID NO:87, or a pharmaceutically acceptable saltthereof.
 3. The modified antisense compound or pharmaceuticallyacceptable salt thereof of claim 1, comprising at least onephosphorothioate intemucleoside linkage.
 4. The modified antisensecompound or pharmaceutically acceptable salt thereof of claim 1,comprising at least one 2′-O-(2-methoxyethyl) sugar moiety.
 5. Themodified antisense compound or pharmaceutically acceptable salt thereofof claim 1, comprising at least one 5-methylcytosine.
 6. The modifiedantisense compound or pharmaceutically acceptable salt thereof of claim1, which is in the form of a sodium salt.
 7. The modified antisensecompound or pharmaceutically acceptable salt thereof of claim 1,comprising the nucleotide sequence shown in SEQ ID NO:87, wherein everyinternucleoside linkage is a phosphorothioate linkage, and wherein inthe nucleotide sequence shown in SEQ ID NO:87, nucleotides 1-4 and 15-18reading from the 5′ end to the 3′ end each comprise a2′-O-(2-methoxyethyl) sugar, nucleotides 5-14 are each2′-deoxynucleotides, and the cytosine residues at nucleotide positions 5and 10 are each 5-methylcytosine.
 8. An antisense compound comprisingthe nucleotide sequence shown in SEQ ID NO:87, wherein everyinternucleoside linkage is a phosphorothioate linkage, and wherein inthe nucleotide sequence shown in SEQ ID NO:87, nucleotides 1-4 and 15-18reading from the 5′ end to the 3′ end each comprise a2′-O-(2-methoxyethyl) sugar, nucleotides 5-14 are each2′-deoxynucleotides, the cytosine residues at nucleotide positions 5 and10 are each 5-methylcytosine, and which is in the form of a sodium salt.9. The modified antisense compound or pharmaceutically acceptable saltthereof of claim 2, comprising at least one phosphorothioateinternucleoside linkage.
 10. The modified antisense compound orpharmaceutically acceptable salt thereof of claim 2, comprising at leastone 2′-O-(2-methoxyethyl) sugar moiety.
 11. The modified antisensecompound or pharmaceutically acceptable salt thereof of claim 2,comprising at least one 5-methylcytosine.
 12. The modified antisensecompound or pharmaceutically acceptable salt thereof of claim 2, whichis in the form of a sodium salt.
 13. The modified antisense compound orpharmaceutically acceptable salt thereof of claim 2, wherein everyinternucleoside linkage is a phosphorothioate linkage, nucleotides 1-4and 15-18 reading from the 5′ end to the 3′ end each comprise a2′-O-(2-methoxyethyl) sugar, nucleotides 5-14 are each2′-deoxy-nucleotides, and the cytosine residues at nucleotide positions5 and 10 are each 5-methylcytosine.
 14. A modified antisense compoundconsisting of the nucleotide sequence shown in SEQ ID NO:87, whereinevery internucleoside linkage is a phosphorothioate linkage, nucleotides1-4 and 15-18 reading from the 5′ end to the 3′ end each comprise a2′-O-(2-methoxyethyl) sugar, nucleotides 5-14 are each2′-deoxy-nucleotides, the cytosine residues at nucleotide positions 5and 10 are each 5-methylcytosine, and which is in the form of a sodiumsalt.
 15. A pharmaceutical composition, comprising said sodium salt ofsaid modified antisense compound of claim 14, and a pharmaceuticallyacceptable carrier, diluent, or excipient.
 16. The pharmaceuticalcomposition of claim 15, further comprising one or more otherchemotherapeutic agent which functions by a non-antisense mechanism. 17.A method of treating a condition or disease associated with Survivinexpression in an animal, comprising administering to said animal aneffective amount of said sodium salt of said modified antisense compoundof claim
 14. 18. The method of claim 17, wherein said condition ordisease associated with Survivin expression is a hyperproliferativecondition or disease.
 19. The method of claim 18, wherein saidhyperproliferative condition or disease is a cancer.
 20. The method ofclaim 19, wherein said cancer is selected from the group consisting oflung cancer, colon cancer, pancreatic cancer, prostate cancer, breastcancer, stomach cancer, non-Hodgkin's lymphoma, neuroblastoma, bladdercancer, and cancer involving keratinocyte or fibroblast cells.
 21. Themethod of claim 17, wherein said animal is a human.
 22. A method oftreating a cancer selected from the group consisting of lung cancer,colon cancer, pancreatic cancer, prostate cancer, breast cancer, stomachcancer, non-Hodgkin's lymphoma, neuroblastoma, bladder cancer, andcancer involving keratinocyte or fibroblast cells in a human, comprisingadministering to said human an effective amount of a modified antisensecompound consisting of the nucleotide sequence shown in SEQ ID NO:87,wherein every internucleoside linkage is a phosphorothioate linkage,nucleotides 1-4 and 15-18 reading from the 5′ end to the 3′ end eachcomprise a 2′-O-(2-methoxyethyl) sugar, nucleotides 5-14 are each2′-deoxy-nucleotides, the cytosine residues at nucleotide positions 5and 10 are each 5-methylcytosine, and which is in the form of a sodiumsalt.
 23. A modified antisense compound consisting of the nucleotidesequence shown in SEQ ID NO:87, wherein every intemucleoside linkage isa phosphorothioate linkage, nucleotides 1-4 and 15-18 reading from the5′ end to the 3′ end each comprise a 2′-O-(2-methoxyethyl) sugar,nucleotides 5-14 are each 2′-deoxynucleotides, at least one cytosine isa 5-methylcytosine, and which is in the form of a sodium salt.
 24. Apharmaceutical composition, comprising said sodium salt of said modifiedantisense compound of claim 23, and a pharmaceutically acceptablecarrier, diluent, or excipient.
 25. A method of treating a cancer in ahuman, comprising administering to said human an effective amount ofsaid sodium salt of said modified antisense compound of claim
 23. 26.The method of claim 25, wherein said cancer is selected from the groupconsisting of lung cancer, colon cancer, pancreatic cancer, prostatecancer, breast cancer, stomach cancer, non-Hodgkin's lymphoma,neuroblastoma, bladder cancer, and cancer involving keratinocyte orfibroblast cells.
 27. A modified antisense compound consisting of thenucleotide sequence shown in SEQ ID NO:87, wherein every intemucleosidelinkage is a phosphorothioate linkage, nucleotides 1-4 and 15-18 readingfrom the 5′ end to the 3′ end each comprise a 2′-O-(2-methoxyethyl)sugar, nucleotides 5-14 are each 2′-deoxynucleotides, and which is inthe form of a sodium salt.
 28. A pharmaceutical composition, comprisingsaid sodium salt of said modified antisense compound of claim 27, and apharmaceutically acceptable carrier, diluent, or excipient.
 29. A methodof treating a cancer in a human, comprising administering to said humanan effective amount of said sodium salt of said modified antisensecompound of claim
 27. 30. The method of claim 29, wherein said cancer isselected from the group consisting of lung cancer, colon cancer,pancreatic cancer, prostate cancer, breast cancer, stomach cancer,non-Hodgkin's lymphoma, neuroblastoma, bladder cancer, and cancerinvolving keratinocyte or fibroblast cells.